Phrenic Nerve Amplitude Research Articles

Cervical spinal demyelination with ethidium bromide impairs respiratory (phrenic) activity and forelimb motor behavior in rats

Although respiratory complications are a major cause of morbidity/mortality in many neural injuries or diseases, little is known concerning mechanisms whereby deficient myelin impairs breathing, or how patients compensate for such changes. Here, we tested the hypothesis that respiratory and forelimb motor functions are impaired in a rat model of focal dorsolateral spinal demyelination (ethidium bromide, EB). Ventilation, phrenic nerve activity and horizontal ladder walking were performed 7–14days post-C2 injection of EB or vehicle (SHAM). EB caused dorsolateral demyelination at C2–C3 followed by significant spontaneous remyelination at 14days post-EB. Although ventilation did not differ between groups, ipsilateral integrated phrenic nerve burst amplitude was significantly reduced versus SHAM during chemoreceptor activation at 7days post-EB but recovered by 14days. The ratio of ipsi- to contralateral phrenic nerve amplitude correlated with cross-sectional lesion area. This ratio was significantly reduced 7days post-EB versus SHAM during baseline conditions, and versus SHAM and 14-day groups during chemoreceptor activation. Limb function ipsilateral to EB was impaired 7days post-EB and partially recovered by 14days post-EB. EB provides a reversible model of focal, spinal demyelination, and may be a useful model to study mechanisms of functional impairment and recovery via motor plasticity, or the efficacy of new therapeutic interventions to reduce severity or duration of disease.

P2Y1‐receptors are expressed by CO2/H+‐insensitive neurons in the retrotrapezoid nucleus (RTN) and contribute to the peripheral drive to breathe

The chemical drive to breathe comes from central chemoreceptors that sense tissue CO2/H+, and peripheral chemoreceptors that sense arterial CO2/H+ and O2. Changes in CO2/H+ and O2 can trigger ATP release near the RTN, and ATP has been shown to modulate activity of RTN neurons, possibly by activation of P2Y1‐receptors (P2Y1‐R). Therefore, we hypothesize that P2Y1‐R represent the molecular basis for central and peripheral chemoreceptor integration at the level of the RTN. Here, we use in vivo and in vitro recording techniques to examine the role of P2Y1‐R expressed by RTN neurons in central and peripheral chemoreception. In vivo, bilateral injection of MRS2365, a P2Y1‐R specific agonist, into the RTN of anesthetized adult rats increased phrenic nerve amplitude 24% and frequency 31%, whereas application of the specific P2Y1‐R antagonist MRS2179 decreased the ventilatory response to peripheral chemoreceptor activation but not hypercapnia. In vitro, loose‐patch recordings of neurons in slices from rats (P7–12) show that baseline activity and CO2/H+‐sensitivity of chemoreceptors was unaffected by MRS2179 or MRS2365. Conversely, MRS2179 decreased baseline activity of non‐chemosensitive RTN neurons by 42 % and MRS2365 increased activity of these cells by 630 %. These results indicate that P2Y1‐R are functionally expressed in nonchemosensitive RTN neurons and contribute to peripheral chemoreception.

Differential Cardiorespiratory and Sympathetic Reflex Responses to Microinjection of Neuromedin U in Rat Rostral Ventrolateral Medulla

The rostral ventrolateral medulla (RVLM) regulates sympathetic vasomotor outflow and reflexes. Intracerebroventricular neuromedin U (NMU) increases sympathetic nerve activity (SNA), mean arterial pressure (MAP), and heart rate (HR), but the central nuclei that mediate these effects are unknown. In urethane-anesthetized, vagotomized, and artificially ventilated male Sprague-Dawley rats (n = 36) the effects of bilateral microinjection of NMU (50 nl, each side) into RVLM on cardiorespiratory variables, somatosympathetic reflex, arterial baroreflex, and chemoreflex were investigated. Microinjection of NMU into RVLM elicited a hypertension, tachycardia, and an increase in splanchnic SNA (SSNA) and lumbar SNA (LSNA) at lower doses (25 and 50 pmol). At higher dose (100 pmol), NMU caused a biphasic response, a brief hypertension and sympathoexcitation followed by prolonged hypotension and sympathoinhibition. The peak excitatory and inhibitory response was found at 100 pmol NMU with an increase in MAP, HR, SSNA, and LSNA of 36 mm Hg, 20 beats per minute, 34%, and 89%, respectively, and a decrease of 33 mm Hg, 25 beats per minute, 42%, and 52%, respectively, from baseline. NMU, in the RVLM, also increased phrenic nerve amplitude and the expiratory period and reduced the inspiratory period. NMU (100 pmol) attenuated the somatosympathetic reflex and the sympathoexcitatory and respiratory responses to hypoxia and hypercapnia. After NMU injection in RVLM, the maximum gain of the SSNA baroreflex function curve was increased, but that of the LSNA was reduced. The present study provides functional evidence for a complex differential modulatory activity of NMU on the cardiovascular and reflex responses that are integrated in the RVLM.

Acute intermittent hypoxia-induced expression of brain-derived neurotrophic factor is disrupted in the brainstem of methyl-CpG-binding protein 2 null mice

Rett syndrome is a neurodevelopmental disorder caused by loss-of-function mutations in the gene encoding the transcription factor methyl-CpG-binding protein 2 (MeCP2). One of its targets is the gene encoding brain-derived neurotrophic factor (bdnf). In vitro studies using cultured neurons have produced conflicting results with respect to the role of MeCP2 in BDNF expression. Acute intermittent hypoxia (AIH) induces plasticity in the respiratory system characterized by long-term facilitation of phrenic nerve amplitude. This paradigm induces an increase in BDNF protein. We hypothesized that AIH leads to augmentation of BDNF transcription in respiratory-related areas of the brainstem and that MeCP2 is necessary for this process. Wild-type and mecp2 null (mecp2−/y) mice were subjected to three 5-min episodes of exposure to 8% O2/4% CO2/88% N2, delivered at 5-min intervals. Normoxia control wild-type and mecp2 null mice were exposed to room air for the total length of time, that is, 30 min. Following a recovery in room air, the pons and medulla were rapidly removed. Expression of BDNF protein and transcripts were determined by ELISA and quantitative PCR, respectively. AIH induced a significant increase in BDNF protein in the pons and medulla, and in mRNA transcript levels in the pons of wild-type animals. In contrast, there were no significant changes in either BDNF protein or transcripts in the pons or medulla of mice lacking MeCP2. The results indicate that MeCP2 is required for regulation of BDNF expression by acute intermittent hypoxia in vivo.

Catestatin, a chromogranin A-derived peptide, is sympathoinhibitory and attenuates sympathetic barosensitivity and the chemoreflex in rat CVLM

Hypertension is a major cause of morbidity. The neuropeptide catestatin [human chromogranin A-(352-372)] is a peptide product of the vesicular protein chromogranin A. Studies in the periphery and in vitro studies show that catestatin blocks nicotine-stimulated catecholamine release and interacts with β-adrenoceptors and histamine receptors. Catestatin immunoreactivity is present in the rostral ventrolateral medulla (RVLM), a key site for blood pressure control in the brain stem. Recently, we reported that microinjection of catestatin into the RVLM is sympathoexcitatory and increases barosensitivity. Here, we report the effects of microinjection of catestatin (1 mM, 50 nl) into the caudal ventrolateral medulla (CVLM) in urethane-anesthetized, bilaterally vagotomized, artificially ventilated Sprague-Dawley rats (n = 8). We recorded resting arterial pressure, splanchnic sympathetic nerve activity, phrenic nerve activity, heart rate, and measured cardiovascular homeostatic reflexes. Homeostatic reflexes were evaluated by measuring cardiovascular responses to carotid baroreceptor and peripheral chemoreceptor activation. Catestatin decreased basal levels of arterial pressure (-23 ± 4 mmHg), sympathetic nerve activity (-26.6 ± 5.7%), heart rate (-19 ± 5 bpm), and phrenic nerve amplitude (-16.8 ± 3.3%). Catestatin caused a 15% decrease in phrenic inspiratory period (T(i)) and a 16% increase in phrenic expiratory period (T(e)) but had no net effect on the phrenic interburst interval (T(tot)). Catestatin decreased sympathetic barosensitivity by 63.6% and attenuated the peripheral chemoreflex (sympathetic nerve response to brief hypoxia; range decreased 39.9%; slope decreased 30.1%). The results suggest that catestatin plays an important role in central cardiorespiratory control.

Vasostatin I (CgA17–76) vasoconstricts rat splanchnic vascular bed but does not affect central cardiovascular function

Vasostatin I (CgA1–76) is a naturally occurring biologically active peptide derived from chromogranin A (CgA), and is so named for its inhibitory effects on vascular tension. CgA mRNA is expressed abundantly in sympathoexcitatory catecholaminergic neurons of the rostral ventrolateral medulla (RVLM). CgA microinjection into the RVLM decreases blood pressure (BP), heart rate (HR) and sympathetic nerve activity (SNA). Proteolytic fragments of CgA are thought to be responsible for the cardiovascular effects observed. We hypothesised that vasostatin I is one of the fragments responsible for the central effects of CgA. We examined the role of a vasostatin I fragment, CgA17–76 (VS-I(CgA17–76)), containing the portion important for biological effects. The effects of VS-I(CgA17–76) delivered by intrathecal injection, or microinjection into the RVLM, on cardio-respiratory function in urethane anaesthetised, vagotomised, mechanically ventilated Sprague-Dawley rats (n=21) were evaluated. The effects of intrathecal VS-I(CgA17–76) on the somato-sympathetic, baroreceptor and peripheral chemoreceptor reflexes were also examined. At the concentrations used (10, 100 or 200μM, intrathecal; or 5μM, RVLM microinjection) VS-I(CgA17–76) produced no change in mean arterial pressure, HR, splanchnic SNA, phrenic nerve amplitude or phrenic nerve frequency. All reflexes examined were unchanged following intrathecal VS-I(CgA17–76). In the periphery, VS-I(CgA17–76) potentiated the contractile effects of noradrenaline on rat mesenteric arteries (n=6), with a significant left-shift in the dose response curve to noradrenaline (3.7×10−7 vs 7.7×10−7). Our results indicate that VS-I(CgA17–76) is active in the periphery but not centrally, and is not a central modulator of cardiorespiratory function and physiological reflexes.

Intrathecal neuromedin U induces biphasic effects on sympathetic vasomotor tone, increases respiratory drive and attenuates sympathetic reflexes in rat

Neuromedin U (NMU) is a brain-gut peptide that plays regulatory roles in feeding, anxiety, smooth muscle contraction, blood flow, pain and adrenocortical function via two receptors, the NMU receptor 1 and NMU receptor 2. NMU has several known functions in the periphery, but its role in central cardiorespiratory regulation remains poorly understood. Experiments were conducted on urethane-anaesthetized, vagotomized and artificially ventilated male Sprague-Dawley rats (n= 42) to determine if NMU modulates sympathetic vasomotor output at the spinal level or modulates baro-, chemo- and somato-sympathetic reflexes. Intrathecal (i.t.) injections of NMU (2.5-20 nmol) caused a dose-dependent biphasic response, initially a brief period of hypertension and sympatho-excitation followed by prolonged hypotension and sympatho-inhibition. Peak excitatory as well as inhibitory responses were observed at 20 nmol. NMU (20 nmol) initially increased mean arterial pressure and splanchnic sympathetic nerve activity by 24 mmHg and 27% and then reduced these by 37 mmHg and 47%, respectively. NMU also dose-dependently increased respiratory drive, as indicated by a rise in phrenic nerve amplitude, an increase in neural minute ventilation and a shortening of the inspiratory period. Both sympatho-excitatory peaks of the somato-sympathetic reflex were abolished by i.t. NMU. Pressor, sympatho-excitatory and tachycardiac responses to chemoreceptor activation (100% N₂) were blocked or significantly reduced following i.t. NMU. NMU also reduced barosensitivity. The data demonstrate that NMU, acting in the spinal cord, differentially contributes to the control of sympathetic tone and adaptive sympathetic reflexes.

Intrathecal bombesin is sympathoexcitatory and pressor in rat

Bombesin, a 14 amino-acid peptide, is pressor when administered intravenously in rat and pressor and sympathoexcitatory when applied intracerebroventricularly. To determine the spinal effects of bombesin, the peptide was administered acutely in the intrathecal space at around thoracic spinal cord level six of urethane-anesthetized, paralyzed, and bilaterally vagotomized rats. Blood pressure, heart rate, splanchnic sympathetic nerve activity (sSNA), phrenic nerve activity, and end-tidal CO(2) were monitored to evaluate changes in the cardiorespiratory systems. Bombesin elicited a long-lasting excitation of sSNA associated with an increase in blood pressure and tachycardia. There was a mean increase in arterial blood pressure of 52 ± 5 mmHg (300 μM; P < 0.01). Heart rate and sSNA also increased by 40 ± 4 beats/min (P < 0.01) and 162 ± 33% (P < 0.01), respectively. Phrenic nerve amplitude (PNamp, 73 ± 8%, P < 0.01) and phrenic expiratory period (+0.16 ± 0.02 s, P < 0.05) increased following 300 μM bombesin. The gain of the sympathetic baroreflex increased from -2.8 ± 0.7 to -5.4 ± 0.9% (P < 0.01), whereas the sSNA range was increased by 99 ± 26% (P < 0.01). During hyperoxic hypercapnia (10% CO(2) in O(2), 90 s), bombesin potentiated the responses in heart rate (-25 ± 5 beats/min, P < 0.01) and sSNA (+136 ± 29%, P < 0.001) but reduced PNamp (from 58 ± 6 to 39 ± 7%, P < 0.05). Finally, ICI-216,140 (1 mM), an in vivo antagonist for the bombesin receptor 2, attenuated the effects of 300 μM bombesin on blood pressure (21 ± 7 mmHg, P < 0.01). We conclude that bombesin is sympathoexcitatory at thoracic spinal segments. The effect on phrenic nerve activity may the result of spinobulbar pathways and activation of local motoneuronal pools.

Intrathecal orexin A increases sympathetic outflow and respiratory drive, enhances baroreflex sensitivity and blocks the somato‐sympathetic reflex

Intrathecal (i.t.) injection of orexin A (OX-A) increases blood pressure and heart rate (HR), but the effects of OX-A on sympathetic and phrenic, nerve activity, and the baroreflex(es), somato-sympathetic and hypoxic chemoreflex(es) are unknown. Urethane-anaesthetized, vagotomized and artificially ventilated male Sprague-Dawley rats were examined in this study. The effects of i.t. OX-A (20 nmol 10 µL⁻¹) on cardiorespiratory parameters, and responses to stimulation of the sciatic nerve (electrical), arterial baroreceptors (phenylephrine hydrochloride, 0.01 mg kg⁻¹ i.v.) and peripheral (hypoxia) chemoreceptors were also investigated. i.t. OX-A caused a prolonged dose-dependent sympathoexcitation, pressor response and tachycardia. The peak effect was observed at 20 nmol with increases in mean arterial pressure, HR and splanchnic sympathetic nerve activity (sSNA) of 32 mmHg, 52 beats per minute and 100% from baseline respectively. OX-A also dose-dependently increased respiratory drive, as indicated by a rise in phrenic nerve amplitude and a fall in phrenic nerve frequency, an increase in neural minute ventilation, a lengthening of the expiratory period, and a shortening of the inspiratory period. All effects of OX-A (20 nmol) were attenuated by the orexin receptor 1 antagonist SB 334867. OX-A significantly reduced both sympathoexcitatory peaks of somato-sympathetic reflex while increasing baroreflex sensitivity. OX-A increased the amplitude of the pressor response and markedly amplified the effect of hypoxia on sSNA. Thus, activation of OX receptors in rat spinal cord alters cardiorespiratory function and differentially modulates sympathetic reflexes.

Somatostatin selectively ablates post-inspiratory activity after injection into the Bötzinger complex

Somatostatin (SST) neurons in the ventral respiratory column (VRC) are essential for the generation of normal breathing. Little is known about the neuromodulatory role of SST on ventral respiratory neurons other than that local administration induces apnoea. Here, we describe the cardiorespiratory effects of microinjecting SST into the preBötzinger and Bötzinger complexes which together elaborate a normal inspiratory augmenting and expiratory respiratory pattern, and on spinally projecting respiratory subnuclei (rostral ventral respiratory group; rVRG). Microinjections (20–50 nl) of SST (0.15, 0.45, 1.5 mM) were made into respiratory subnuclei of urethane-anaesthetized, paralysed, vagotomized and artificially ventilated Sprague–Dawley rats (n=46). Unilateral microinjection of SST into the Bötzinger complex converted the augmenting activity of phrenic nerve discharge into a square-wave apneustic pattern associated with a lengthening of inspiratory period and shortening of expiratory time. Following bilateral microinjection the apneusis became pronounced and was associated with a dramatic variability in inspiratory duration. Microinjection of SST into the Bötzinger complex also abolished the post-inspiratory (post-I) motor activity normally observed in vagal and sympathetic nerves. In the preBötzinger complex SST caused bradypnoea and with increasing dose, apnoea. In the rVRG SST reduced phrenic nerve amplitude, eventually causing apnoea. In conclusion, SST powerfully inhibits respiratory neurons throughout the VRC. Of particular interest is the finding that chemical inhibition of the Bötzinger complex with SST ablates the post-I activity that is normally seen in respiratory activity and leads to apneusis. This loss of post-I activity is a unique feature of inhibition with SST and is not seen following inhibition with other agents such as galanin, GABA and endomorphin. The effect seen on post-I activity is similar to the effect of inhibiting the Kölliker–Fuse nucleus in the pons. The mechanism by which SST exerts this effect on Bötzinger neurons remains to be determined.

Microinjection of methysergide into the raphe nucleus attenuated phrenic long-term facilitation in rats

Exposure to acute intermittent hypoxia (AIH) evokes persistent increase in respiratory activity that lasts up to 60 min after hypoxic episodes have ceased. This persistent increase in phrenic nerve activity (PNA) is known as phrenic long-term facilitation (LTF). AIH-induced phrenic LTF in anesthetized rats is serotonin dependant. The present study was performed to determine whether microinjection of methysergide (4 mM, 20 +/- 5 nl), a broad spectrum 5-HT receptor antagonist, into the caudal raphe nuclei influences phrenic LTF. Peak integrated PNA and respiratory frequency were recorded at 15, 30, and 60 min after five 3-min episodes of normocapnic hypoxia in urethane-anesthetized, vagotomized, paralyzed and ventilated male Sprague-Dawley rats. In control animals, phrenic nerve amplitude was elevated 66.7 +/- 8.6% from baseline 1 h after episodic hypoxia, indicating phrenic LTF. Experimental microinjections of methysergide prior to AIH exposure attenuated phrenic LTF (amplitude increase 2.62 +/- 2.9% over baseline). We conclude that methysergide microinjections into the caudal raphe region attenuated phrenic LTF induced by AIH, indicating involvement of 5-HT receptor activation at a supraspinal level.

Association of paraspinal and diaphragm denervation in ALS

Paraspinal EMG needle examination is commonly performed in amyotrophic lateral sclerosis (ALS) for diagnosis. Because lower motor neurons for axial muscles and diaphragm are located medially in the anterior horn, we tested if involvement of axial muscles is associated with diaphragm weakness in ALS. Forty-four ALS patients were included with ALSFRS greater than 20/40. We used needle EMG to search for signs of denervation in biceps, tibialis anterior, C6 and T5 paraspinal muscles, and intercostal and diaphragm muscles. We also evaluated phrenic nerve motor responses and forced vital capacity (FVC). We tested specificity, sensitivity, and discriminative strength (ROC analysis). Fibs-sw in C6 and T5 paraspinal muscles, as well as fibs-sw in diaphragm and intercostal muscles showed high specificity and positive predictive value for FVC<80%. Discriminative strength was good for all the above tests, as well as for phrenic nerve amplitude and ALSFRS regarding FVC<80%. Axial muscles denervation is related to diaphragm denervation and therefore to poor respiratory function in ALS. We suggest that medially located lower motor neurons are affected concurrently in ALS.

Chronic hypoxia decreases response to central chemoreceptor stimulation in the nucleus tractus solitarius (NTS)

One hallmark of acclimatization to chronic hypoxia (CH) is hyperventilation and a lower arterial carbon dioxide (CO2) level. These effects persist upon return to normoxia, suggesting a change in the regulation of CO2 during CH. We tested the hypothesis that plasticity in one central chemoreceptor site, the NTS, contributes to altered CO2 regulation in CH. In urethane‐anesthetized rats, we stimulated chemoreceptors in the NTS by a unilateral microinjection of acetazolamide (ACZ, 1 nl of 0.3 μM), to produce focal acidosis. The product of rectified, integrated phrenic nerve amplitude and frequency (Phr) was measured 30 min after ACZ in normoxic control (N; n=5) and CH rats (n=5). ACZ increased Phr significantly more in N rats than CH rats (N=263±14%; CH=83±18%; p&lt;0.001). ACZ in adjacent brainstem regions had no effect on Phr. All rats showed the capacity to increase Phr to a 10% inspired CO2 challenge following ACZ, suggesting that the dose of ACZ did not produce a maximal phrenic response. These results indicate plasticity in chemosensitivity in the NTS but they cannot easily explain the lower CO2 set point with CH.Supported by NIH HL81823 (FLP) and AHA 0615069Y (KAW).

Neuropeptide Y in the rostral ventrolateral medulla blocks somatosympathetic reflexes in anesthetized rats

The rostral ventrolateral medulla (RVLM) is a major integrative center of cardiovascular reflexes that modulate vasomotor tone. The functions of Neuropeptide Y (NPY) in the RVLM on cardiorespiratory responses remain unknown. Arterial blood pressure (AP), heart rate (HR), splanchnic sympathetic (sSNA) and phrenic nerve activities, and responsiveness to baro-, somatosympathetic, and chemoreflex stimulation were recorded before and after bilateral NPY injection (100 pmol, 200 nl/side) in the RVLM of vagotomized urethane-anesthetized rats ( n = 7). Responses were characterized by an initial increase in AP followed by prolonged hypotension ( P < 0.01). A similar biphasic effect was exerted on HR ( P < 0.01). NPY caused a large increase of sSNA ( P < 0.01), that gradually recovered towards baseline. Somatosympathetic responses evoked by tibial nerve stimulation were largely abolished following NPY microinjection ( P < 0.01), but sympathoexcitatory responses evoked by acute hypoxia or sympathoinhibition evoked by aortic depressor nerve stimulation were unchanged following NPY. There was no effect of NPY on phrenic nerve amplitude or frequency. We conclude that NPY exerts excitatory effects on sympathetic tone, but inhibits responses evoked by somatic inputs. We speculate that this apparent contradiction may be due to differential expression of NPY receptor subtypes on the soma of sympathetic premotor neurons in the RVLM and on the presynaptic terminals of neurons that comprise excitatory afferent pathways.

Chronic hypoxia abolishes expiratory prolongation following carotid sinus nerve stimulation in the anesthetized rat

In anesthetized rats, increases in phrenic nerve (PN) amplitude and frequency during brief periods of hypoxia or electrical stimulation of the carotid sinus nerve (CSN) are followed by an increase in expiratory duration. We investigated the effects of chronic exposure to hypoxia on PN responses to CSN stimulation. In Inactin anesthetized (100 mg/kg) Sprague-Dawley rats PN discharge and arterial pressure responses to 10-120 s of CSN stimulation (20 Hz, 0.2 ms duration pulses) were recorded after 7-10 days exposure to hypoxia (10 +/- .5% O2). In normoxic rats, the degree of CSN-evoked expiratory prolongation was dependent upon the duration of CSN stimulation. CSN-evoked increases in PN burst amplitude were not different comparing chronic hypoxic rats to rats maintained at normoxia while CSN-evoked increases in PN burst frequency were greater in chronic hypoxic rats (p<.05). CSN-evoked expiratory prolongation was abolished in chronic hypoxic rats. Following chronic hypoxia, changes occur within the central processing of arterial chemoreceptor inputs so that CSN stimulation evokes an enhanced PN frequency response and no expiratory prolongation.

A mapping study of cardiorespiratory responses to chemical stimulation of the midline medulla oblongata in ventilated and freely breathing rats.

The aim of this study was to examine the cardiorespiratory effects of chemically stimulating neurons in the midline medulla oblongata (MM) of artificially ventilated and freely breathing anesthetized rats. Earlier studies reported that stimulation of the MM elicits increases or decreases in mean arterial pressure (MAP) and phrenic nerve activity, depending on the mode and site of stimulation, anesthetic, and species. In the first series of experiments, rats were anesthetized with urethane, artificially ventilated, paralyzed, and bilaterally vagotomized. The rostrocaudal extent of the MM was mapped by microinjections of DL-homocysteic acid or L-glutamate (both 100 mM, 100 nl), and, in line with previous studies, most injections produced only small responses in MAP, heart rate, and splanchnic sympathetic nerve activity. Increases in respiratory parameters were evoked in caudal regions. However, activation of a discrete region of the MM at the level of the caudal pole of the facial nucleus (CP7) consistently caused a dramatic reduction in phrenic nerve amplitude and/or frequency and, in six rats, produced a prolonged apnea. The second series of experiments was carried out on freely breathing pentobarbitone sodium-anesthetized rats, with a diaphragmatic electromyogram used to monitor respiratory activity. Respiratory activity could again be abolished at CP7 after microinjections of glutamate (100 mM, 50 nl); however, these responses were accompanied by large decreases in MAP and moderate reductions in heart rate. This depression of respiratory activity may be due to activation of propriobulbar inhibitory neurons that project to known respiratory centers in the brain stem.

Electrophysiological evaluation of phrenic nerve injury during cardiac surgery – a prospective, controlled, clinical study

BackgroundAccording to some reports, left hemidiaphragmatic paralysis due to phrenic nerve injury may occur following cardiac surgery. The purpose of this study was to document the effects on phrenic nerve injury of whole body hypothermia, use of ice-slush around the heart and mammary artery harvesting.MethodsElectrophysiology of phrenic nerves was studied bilaterally in 78 subjects before and three weeks after cardiac or peripheral vascular surgery. In 49 patients, coronary artery bypass grafting (CABG) and heart valve replacement with moderate hypothermic (mean 28°C) cardiopulmonary bypass (CPB) were performed. In the other 29, CABG with beating heart was performed, or, in several cases, peripheral vascular surgery with normothermia.ResultsIn all patients, measurements of bilateral phrenic nerve function were within normal limits before surgery. Three weeks after surgery, left phrenic nerve function was absent in five patients in the CPB and hypothermia group (3 in CABG and 2 in valve replacement). No phrenic nerve dysfunction was observed after surgery in the CABG with beating heart (no CPB) or the peripheral vascular groups. Except in the five patients with left phrenic nerve paralysis, mean phrenic nerve conduction latency time (ms) and amplitude (mV) did not differ statistically before and after surgery in either group (p > 0.05).ConclusionsOur results indicate that CPB with hypothermia and local ice-slush application around the heart play a role in phrenic nerve injury following cardiac surgery. Furthermore, phrenic nerve injury during cardiac surgery occurred in 10.2 % of our patients (CABG with CPB plus valve surgery).

Chronic hypoxia abolishes posthypoxia frequency decline in the anesthetized rat.

In anesthetized rats, increases in phrenic nerve amplitude and frequency during brief periods of hypoxia are followed by a reduction in phrenic nerve burst frequency [posthypoxia frequency decline (PHFD)]. We investigated the effects of chronic exposure to hypoxia on PHFD and on peripheral and central O2-sensing mechanisms. In Inactin-anesthetized (100 mg/kg) Sprague-Dawley rats, phrenic nerve discharge and arterial pressure responses to 10 s N2 inhalation were recorded after exposure to hypoxia (10 +/- 0.5% O2) for 6-14 days. Compared with rats maintained at normoxia, PHFD was abolished in chronic hypoxic rats. Because of inhibition of PHFD, the increased phrenic burst frequency and amplitude after N2 inhalation persisted for 1.8-2.8 times longer in chronic hypoxic (70 s) compared with normoxic (25-40 s) rats (P < 0.05). After acute bilateral carotid body denervation, N2 inhalation produced a short depression of phrenic nerve discharge in both chronic hypoxic and normoxic rats. However, the degree and duration of depression of phrenic nerve discharge was smaller in chronic hypoxic compared with normoxic rats (P < 0.05). We conclude that after exposure to chronic hypoxia, a reduction in PHFD contributes to an increased duration of the acute hypoxic ventilatory response in anesthetized rats. Furthermore, after exposure to chronic hypoxia, the central network responsible for respiration is more resistant to the depressant effects of acute hypoxia in anesthetized rats.

Presynaptic Δ opioid receptors differentially modulate rhythm and pattern generation in the ventral respiratory group of the rat

The specific role of the Δ opioid receptor (DOR), in opioid-induced respiratory depression in the ventral respiratory group (VRG) is largely unknown. Here, we sought to determine (1) the relationship between DOR-immunoreactive (ir) boutons, bulbospinal and functionally identified respiratory neurons in the VRG and (2) the effects of microinjection of the selective DOR agonist, d-Pen 2,5-enkephalin (DPDPE), into different subdivisions of the VRG, on phrenic nerve discharge and mean arterial pressure. Following injections of retrograde tracer into the spinal cord or intracellular labelling of respiratory neurons, in Sprague-Dawley rats, brainstem sections were processed for retrograde or intracellular labelling and DOR-ir. Bulbospinal neurons were apposed by DOR-ir boutons regardless of whether they projected to single (cervical or thoracic ventral horn) or multiple (cervical and thoracic ventral horn) targets in the spinal cord. In the VRG, a total of 24±5% (67±13/223±49) of neurons projecting to the cervical ventral horn, and 37±3% (96±22/255±37) of neurons projecting to the thoracic ventral horn, received close appositions from DOR-ir boutons. Furthermore, DOR-ir boutons closely apposed six of seven intracellularly labelled neurons, whilst the remaining neuron itself possessed boutons that were DOR-ir. DPDPE was microinjected (10 mM, 60 nl, unilateral) into regions of respiratory field activity in the VRG of anaesthetised, vagotomised rats, and the effects on phrenic nerve discharge and mean arterial pressure were recorded. DPDPE depressed phrenic nerve amplitude, with little effect on phrenic nerve frequency in the Bötzinger complex, pre-Bötzinger complex and rVRG, the greatest effects occurring in the Bötzinger complex. The results indicate that the DOR is located on afferent inputs to respiratory neurons in the VRG. Activation of the DOR in the VRG is likely to inhibit the release of neurotransmitters from afferent inputs that modulate the pattern of activity of VRG neurons.

Mu opioid receptors in rat ventral medulla: effects of endomorphin-1 on phrenic nerve activity

Anatomical and in vitro studies suggest that mu opioid receptors (MOR) on pre-Bötzinger complex neurons are responsible for opioid induced respiratory depression (Grey et al., Science 286 (1999) 1566). However, mu opioid agonists injected in vivo, in other regions of the ventral respiratory group (VRG), produce respiratory depression, suggesting that opioids are widely distributed in the VRG. We therefore re-examined the distribution of the MOR in the ventral medulla and found MOR-immunoreactive neurons and terminals in all subdivisions of the VRG. Furthermore, we determined, in rats, the effects of a MOR agonist (endomorphin-1, 10 mM, 60 nl, unilateral), microinjected into different subdivisions of the VRG, on phrenic nerve activity. Endomorphin-1 produced changes in phrenic nerve frequency and amplitude, throughout the VRG. Unexpectedly, endomorphin-1 microinjected into the Bötzinger and pre-Bötzinger complexes consistently increased phrenic nerve frequency. These results support the widespread distribution of MOR in the VRG and also indicate that endomorphin-1, a postulated endogenous ligand, may differentially regulate respiration.